Neural networks in the brain control sleep and circadian rhythms, key interacting processes that regulate numerous physiological and behavioral outputs. Human disorders caused or exacerbated by impaired regulation of sleep and circadian rhythms-such as narcolepsy, genetic sleep phase disturbances, jet lag, shift work, sleep deprivation, depression, etc-are a major source of morbidity, mortality, and economic hardship. Their amelioration will be facilitated by understanding how sleep and circadian rhythm control circuits function in vivo, importantly including intercellular synaptic signaling and homeostatic plasticity. One of the key features of sleep-wake regulation is the ability to rapidly transition from one state to the other, such as to wake up upon receipt of sensory stimuli signaling danger. Current models of rapid sleep state switching in mammals involve mutually inhibitory feedback loops between sleep-promoting and wake-promoting populations of neurons to implement a bistable flip-flop. Sleep flip-flop and circadian regulatory circuits rely on both classical rapid synaptic signaling, as well as small molecule and peptide neuromodulators. Our long- term goal is mechanistic dissection of synaptic communication, neuromodulation, and their interaction in sleep and circadian control circuits of the intact animal. In pursuit of this goal w combine the cell-specific neurogenetic manipulability of the Drosophila model system with whole-cell patch-clamp and functional imaging. We will combine neurogenetic manipulation of classical synaptic release, optogenetic neuronal stimulation, whole-cell patch-clamp, and fluorescent imaging in vivo of intracellular Ca2+ and membrane potential to analyze the functional relationships within and between the sleep-promoting and wake-promoting neurons of the mushroom body to determine how the mushroom body controls sleep bidirectionally and whether it behaves as a bistable flip-flop.We will combine neurogenetic manipulation of classical synaptic release, optogenetic neuronal stimulation, and whole-cell patch-clamp in intact fly brain to determine the synaptic connections that underlie the functional network. We will also test the hypothesis that one or more of the sleep- and/or wake-promoting mushroom body neuron classes encodes homeostatic sleep drive that biases the network to one or the other state.